AMP-activated protein kinase (AMPK) coordinates metabolism with energy availability in eukaryotes by responding to changes in intracellular ATP and AMP levels. The kinase activity of AMPK is stimulated by AMP and inhibited by excess ATP, and it is thought that this unique regulatory behavior enables AMPK to act as a central cellular "fuel gauge". AMPK is thus the subject of intense interest as a target for therapeutics to treat metabolic disorders such as diabetes and obesity. AMPK is an abg heterotrimer that includes a subunit with serine/threonine kinase activity and an adenylate-binding regulatory region composed of elements from all three subunits. In preliminary data for this application we present crystal structures for AMP- and ATP-bound forms of the heterotrimeric adenylate sensor from the Schizosacharomyces pombe enzyme. This complex lacks the kinase catalytic domain, but reveals the conserved trimeric core architecture of AMPKs. ATP and AMP bind competitively to a single site within the g subunit, helping to explain their competing effects. Biophysical experiments show that the adenylate sensor complex binds the a subunit kinase domain in the presence of AMP but ATP binding prevents this association. These data help to provide an initial molecular understanding of AMPK regulation. A crystal structure of an AMPK-ADP complex, surprisingly, reveals a second binding site that can uniquely accommodate ADP. The overarching goal of this application is to gain an atomic-level understanding of AMPK regulation through the following specific aims: (1) characterizes the affinities of binding of various adenylate ligands, and use biophysical methods to determine how ligand binding affects interaction between the regulatory and kinase domains. Results from these studies will be correlated with kinase activity in various ligand-bound states. (2) To gain an understanding of the holoenzyme architecture, we will use site-directed mutagenesis to define the molecular regions responsible for nucleotide-dependent association between the kinase domain and regulatory adenylate sensor. Results from the proposed work will be critical for the rational development of AMPK-directed therapeutics. AMPK, a central regulator of cellular metabolism, is among the most attractive molecular targets for new therapeutics to treat diabetes, obesity, and other metabolic disorders. Prior studies have shown that activators of AMPK administered to diabetic animals can substantially ameliorate the physiological effects of diabetes. Despite the great promise of AMPK-directed therapeutics, little is known about the molecular mechanisms of regulation, and the design of appropriate small molecule drugs has been impeded by the lack of atomic-level information on the architecture of the enzyme. Our preliminary results and the further work proposed will provide high-resolution structural information on AMPK, and should directly enable the rational design of AMPK-directed therapeutics. PUBLIC HEALTH RELEVANCE: AMPK, a central regulator of cellular metabolism, is among the most attractive molecular targets for new therapeutics to treat diabetes, obesity, and other metabolic disorders. Prior studies have shown that activators of AMPK administered to diabetic animals can substantially ameliorate the physiological effects of diabetes. Despite the great promise of AMPK-directed therapeutics, little is known about the molecular mechanisms of regulation, and the design of appropriate small molecule drugs has been impeded by the lack of atomic-level information on the architecture of the enzyme. Our preliminary results and the further work proposed will provide high-resolution structural information on AMPK, and should directly enable the rational design of AMPK-directed therapeutics.